The existence of latent reservoirs of HIV-infected cells constitutes the major impediment towards viral eradication. HIV-1 latent reservoirs are small, but extremely long-lived. Latent infection is associated with undetectable levels of viral gene expression and appears to be non-cytopathic. However, upon reactivation, latent viruses enter an active mode of replication in which they are fully competent for spread and induction of disease. The current thinking in the field is that a combination of hypothetical drugs that will reactivate latent viruses (Latency Reversing Agent or LRA), with present-day antiretroviral drugs, will be an effective approach toward viral eradication. We have previously found that Pam3CSK4, a TLR-1/2 agonist, can reactivate latent HIV-1. Based on this earlier finding, we propose to further investigate the possibility of developing Pathogen Recognition Receptor (PRR) agonists as novel strategies to reactivate/eradicate latent HIV-1 infection. In Aim 1, we plan to characterize the ability of multi-PRR agonists to reactivate latent HIV-1. Multi-PRR agonists are synthetic TLR agonists developed by InvivoGen to be used as adjuvants in cancer therapy, infectious diseases and vaccination. Our preliminary data using the tumoral cell line JLat, a primary model of HIV-1 latency (Cultured TCM model) and cells isolated from aviremic patients demonstrate that these ligands have the ability to reactivate latent HIV-1. Furthermore, in this aim we also plan to evaluate the potential of this agonist to reduce the latent reservoir in vitro. We have found that dynasore, a GTPase inhibitor, has the ability to reactivate latent HIV-1. It has been proposed that dynasore activates the PRR mitochondria antiviral signal protein (MAVS). We have further corroborated this result. Based on this notion, this aim is designed to characterize whether manipulation of the RIG-I/MDA-5/MAVS pathway can have therapeutic implications towards HIV-1 eradication. First, we are going to study whether RIG-1 and MDA-5 ligands that signal through MAVS can reactivate latent HIV-1. Second, it has been shown that the HIV-1 protease can induce lysosomal degradation of RIG-I to avoid sensing and the induction of an interferon response. We plan to characterize whether this mechanism is involved in the establishment of latency. Lastly, identifying novel mechanisms that are involved in the maintenance of latency would lead to the design of novel therapeutic approaches. We have performed a screening to identify epigenetic modulators that synergize with the synthetic TLR-2 ligand CL572. We have identified several pathways that synergize with CL572. In Aim 3, we plan to characterize in detail the mechanism of synergy between PRR agonists and two known pathways, HDACs and bromodomain proteins, and two novel pathways, O6-Methylguanine-DNA methyltransferase and sirtuin-1 and -2. The overall goal of this proposal is to move forward novel targets that can be exploited therapeutically to either eradicate viral reservoirs for HIV-1 infections or to induce a long-term remission without treatment (functional cure).